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A calcified amorphous tumor (CAT) of the heart is a rare, non-neoplastic, intracavitary cardiac mass. Histological examination reveals the presence of calcified and amorphous fibrous material with underlying chronic inflammation. Some studies have reported that CAT typically exhibits rapid growth. However, we observed a case in which CAT unexpectedly ruptured within approximately two weeks. There was no cerebral infarction or significant valvular disease, therefore we were not sure about the indication for surgery; however considering the epidemiological possibility of CAT, we decided to operate and were able to treat the patient without complications., Competing Interests: None declared., (Published by Oxford University Press and JSCR Publishing Ltd. © The Author(s) 2025.)

Dipeptide production mediated by dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11 plays a crucial role in growth of Porphyromonas gingivalis , a periodontopathic asaccharolytic bacterium. Given the particular P1-position specificity of DPPs, it has been speculated that DPP5 or DPP7 might be responsible for degrading refractory P1 amino acids, i.e. , neutral (Thr, His, Gly, Ser, Gln) and hydrophilic (Asn) residues. The present results identified DPP7 as an entity that processes these residues, thus ensuring complete production of nutritional dipeptides in the bacterium. Activity enhancement by the P1' residue was observed in DPP7, as well as DPP4 and DPP5. Toward the refractory P1 residues, DPP7 uniquely hydrolyzed HX|LD-MCA (X = His, Gln, or Asn) and their hydrolysis was most significantly suppressed in dpp7 gene-disrupted cells. Additionally, hydrophobic P2 residue significantly enhanced DPP7 activity toward these substrates. The findings propose a comprehensive 20 P1 × 20 P2 amino acid matrix showing the coordination of four DPPs to achieve complete dipeptide production along with subsidiary peptidases. The present finding of a broad substrate specificity that DPP7 accounts for releasing 48 % (192/400) of N-terminal dipeptides could implicate its potential role in linking periodontopathic disease to related systemic disorders., (© 2025 Walter de Gruyter GmbH, Berlin/Boston.)

A 78-year-old man underwent pericardial patch repair for left ventricular (LV) rupture during mitral valve replacement. After the first operation, a huge (>10 cm) LV pseudoaneurysm was detected, necessitating reoperation. LV rupture is a rare but often fatal complication of mitral valve replacement. Although repair of LV rupture during mitral valve replacement has been reported, the development of pseudoaneurysm after such repair is exceedingly rare. In this case, we successfully treated a huge LV pseudoaneurysm using two pericardial patches to sandwich the rupture hole from the inside., Competing Interests: None declared., (Published by Oxford University Press and JSCR Publishing Ltd. © The Author(s) 2024.)

Isolated left vertebral artery (ILVA) is one of the most frequent vertebral abnormalities. When performing thoracic endovascular aortic repair (TEVAR), the ILVA may have to be closed depending on the position of the stent graft; in these cases, the decision to reconstruct the ILVA depends on the state of cerebral blood flow. Here, we report a case of a 68-year-old male, in whom the Willis arterial circle was incomplete; we therefore performed a reconstructive method during zone 2-landing TEVAR that ensured ILVA and left subclavian artery blood flow without the use of artificial vessels. Only one supraclavicular incision was required for reconstruction. This method has some procedural difficulties; however, it does not use artificial blood vessels and can be performed with a single incision., Competing Interests: None declared., (Published by Oxford University Press and JSCR Publishing Ltd. © The Author(s) 2024.)

Circulating leukocytes enter tissue either through endothelial junctions (paracellular) or via a pore through the body of endothelial cells (transcellular). We have previously shown that genetically replacing VE-cadherin with a VE-cadherin-α-catenin (VEC-αC) fusion construct-which binds constitutively to actin-obstructs junctions, and blocks leukocyte extravasation in lung, skin and postcapillary venules of cremaster muscle. However, neutrophil recruitment into the inflamed peritoneal cavity was unimpaired. Investigating reasons for this, here, we visualized neutrophil diapedesis by 3D intravital video microscopy in the cremaster muscle and omentum, the major site of neutrophil recruitment into the peritoneal cavity. We found that 80% of neutrophil-extravasation occurred through HEVs in the omentum, which was unimpaired by VEC-αC. In addition, in larger venules (60-85 µm) of both tissues, less than 15% of neutrophils extravasated transcellularly in WT mice. However, in VEC-α-C mice, transcellular diapedesis increased severalfold in the omentum, but not in the cremaster. In line with this, omental venules expressed higher levels of ICAM-1 and atypical chemokine receptor 1. Furthermore, only in the omentum, VEC-αC expression caused reduced elongation of venular endothelium in flow-direction, suggesting different biomechanical properties. Collectively, VEC-αC does not inhibit paracellular transmigration in all types of venules and can modulate the diapedesis route., (© 2024. The Author(s).)

Purpose Postoperative infections pose an important problem for patients with cardiac disease. Moreover, oral health status is associated with the risk of longer hospital stays. Therefore, the oral health status of patients was assessed before open-heart surgery. This study aimed to determine the relationship between oral health status and postoperative status. Methods The study included 25 patients who underwent open-heart surgery at our university hospital in 2020. Upon admission, dentists conducted an oral examination and assessed the oral health status of the patients, also using the Japanese version of the Oral Health Assessment Tool (OHAT-J), Revised Oral Assessment Guide (ROAG), oral moisture level, oral bacteria, and other relevant factors. The study investigated the association with postoperative status. Findings Significant postoperative infections were found in patients aged ≥70 years, with an OHAT-J score of ≥5, OHAT-J lip score of ≥1, Streptococcus γ count of 1.0 × 10^6 or higher (CFU/mL), and increased Streptococcus γ before and after surgery. The duration of hospitalization correlated with the OHAT-J, OHAT-J gum and tissue, and ROAG scores. The duration of intensive care unit (ICU) stays correlated with the OHAT-J score. Conclusions The study demonstrates that OHAT-J scores are linked with predicting not just postoperative infection but also the length of hospitalization and ICU stay. As OHAT-J scores do not necessitate specialized dental instruments, they are straightforward and beneficial for healthcare professionals outside of dentistry., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. The Ethics Review Committee for Research in Human Subjects, Showa University Graduate School of Medicine issued approval 3033. The study protocol was performed in accordance with the Declaration of Helsinki. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: This study was funded by the Grants-in-Aid for Scientific Research (KAKENHI) - Scientific Research 18K17294. The authors declare no conflicts of interest associated with this manuscript. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Ono et al.)

This case report details the management of a 79-year-old man who developed massive postoperative pneumothorax following redo coronary artery bypass grafting due to severe lung adhesions. We successfully treated the patient using veno-venous extracorporeal membrane oxygenation without femoral cannulation, allowing for early rehabilitation initiation. Veno-venous extracorporeal membrane oxygenation is a reasonable option for cases of severe respiratory failure due to pneumothorax with lung destruction caused by re-sternotomy during re-do cardiac surgery., Competing Interests: None declared., (Published by Oxford University Press and JSCR Publishing Ltd. © The Author(s) 2024.)

Accumulating evidence suggests that endothelial cells can be useful therapeutic targets. One of the potential targets is an endothelial cell-specific protein, Roundabout4 (ROBO4). ROBO4 has been shown to ameliorate multiple diseases in mice, including infectious diseases and sepsis. However, its mechanisms are not fully understood. In this study, using RNA-seq analysis, we found that ROBO4 downregulates prostaglandin-endoperoxide synthase 2 (PTGS2), which encodes cyclooxygenase-2. Mechanistic analysis reveals that ROBO4 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1) and TNF receptor-associated factor 7 (TRAF7), a ubiquitin E3 ligase. In this complex, ROBO4 enhances IQGAP1 ubiquitination through TRAF7, inhibits prolonged RAC1 activation, and decreases PTGS2 expression in inflammatory endothelial cells. In addition, Robo4-deficiency in mice exacerbates PTGS2-associated inflammatory diseases, including arthritis, edema, and pain. Thus, we reveal the molecular mechanism by which ROBO4 suppresses the inflammatory response and vascular hyperpermeability, highlighting its potential as a promising therapeutic target for inflammatory diseases., (© 2024. The Author(s).)

We recently reported that the activities of dipeptidyl-peptidase (DPP)7 and DPP11, S46-family exopeptidases were significantly elevated by the presence of prime-side amino acid residues of substrates caused by an increase in k cat [Ohara-Nemoto Y. et al., J Biol Chem 298(3):101585. doi: 10.1016/j.jbc.2022]. In the present study, the effects of prime-side residues on Glu-specific endopeptidase I/GluV8 from Staphylococcus aureus were investigated using a two-step cleavage method with tetrapeptidyl-methycoumaryl-7-amide (MCA) carrying P2- to P2'-position residues coupled with DPP11 as the second enzyme. GluV8 showed maximal activity toward benzyloxycarbonyl (Z)-LLE-MCA, while the effects of hydrolysis of substrates one residue shorter, such as acetyl (Ac)-Val-Glu- and Leu-Glu-MCA, were negligible. Nevertheless, activity towards Ac-VE-|-ID-MCA, a substrate carrying P1' and P2' residues, emerged and reached a level 44 % of that for Z-LLE-MCA. Among 11 Ac-HAXD-MCA (X is a varied amino acid), the highest level of activity enhancement was achieved with P1'-Leu and Ile, followed by Phe, Val, Ser, Tyr, and Ala, while Gly and Lys showed scant effects. This activation order was in parallel with the hydrophobicity indexes of these amino acids. The prime-side residues increased k cat /K M primarily through a maximum 500-fold elevation of k cat as well as S46-family exopeptidases. The MEROPS substrate database also indicates a close relationship between activity and hydrophobicity of the P1' residues in 93 N-terminal-truncated substrates, though no correlation was observed among all 4328 GluV8 entities examined. Taken together, these results are the first to demonstrate N-terminal exopeptidase activity of GluV8, considered to be prompted by hydrophobic P1' amino acid residues., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

We report the case of a 76-year-old woman with an incomplete atrioventricular septal defect and severe congestive heart failure who underwent surgical repair. Surgical intervention involved mitral valve repair and patch closure of the ostium primum defect, resulting in a favorable postoperative course. Successful outcomes support surgery as a reasonable treatment option owing to its significant improvement in postoperative quality of life, even in elderly patients with left atrioventricular valve degeneration., Competing Interests: None declared., (Published by Oxford University Press and JSCR Publishing Ltd. © The Author(s) 2024.)

Despite the revolutionary success of chimeric antigen receptor (CAR)-T therapy for hematological malignancies, successful CAR-T therapies for solid tumors remain limited. One major obstacle is the scarcity of tumor-specific cell-surface molecules. One potential solution to overcome this barrier is to utilize antibodies that recognize peptide/major histocompatibility complex (MHCs) in a T cell receptor (TCR)-like fashion, allowing CAR-T cells to recognize intracellular tumor antigens. This study reports a highly specific single-chain variable fragment (scFv) antibody against the MAGE-A4 p230-239 /human leukocyte antigen (HLA)-A∗02:01 complex (MAGE-A4 pMHC), screened from a human scFv phage display library. Indeed, retroviral vectors encoding CAR, utilizing this scFv antibody as a recognition component, efficiently recognized and lysed MAGA-A4 + tumor cells in an HLA-A∗02:01-restricted manner. Additionally, the adoptive transfer of T cells modified by the CAR-containing glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related receptor (GITR) intracellular domain (ICD), but not CD28 or 4-1BB ICD, significantly suppressed the growth of MAGE-A4 + HLA-A∗02:01 + tumors in an immunocompromised mouse model. Of note, a comprehensive analysis revealed that a broad range of amino acid sequences of the MAGE-A4p 230-239 peptide were critical for the recognition of MAGE-A4 pMHC by these CAR-T cells, and no cross-reactivity to analogous peptides was observed. Thus, MAGE-A4-targeted CAR-T therapy using this scFv antibody may be a promising and safe treatment for solid tumors., Competing Interests: Declaration of interests M.M. and Y.K. are employees of Sony Group Corporation, which collaborated in the development of a closed-cell isolation system. The Department of Personalized Cancer Immunotherapy, Mie University Graduate School of Medicine, is an endowment department supported by a grant from T cell Nouveau., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Endothelial cells of mammalian blood vessels have multiple levels of heterogeneity along the vascular tree and among different organs. Further heterogeneity results from blood flow turbulence and variations in shear stress. In the aorta, vascular endothelial protein tyrosine phosphatase (VE-PTP), which dephosphorylates tyrosine kinase receptor Tie2 in the plasma membrane, undergoes downstream polarization and endocytosis in endothelial cells exposed to laminar flow and high shear stress. VE-PTP sequestration promotes Tie2 phosphorylation at tyrosine992 and endothelial barrier tightening. The present study characterized the heterogeneity of VE-PTP polarization, Tie2-pY992 and total Tie2, and claudin-5 in anatomically defined regions of endothelial cells in the mouse descending thoracic aorta, where laminar flow is variable and IgG extravasation is patchy. We discovered that VE-PTP and Tie2-pY992 had mosaic patterns, unlike the uniform distribution of total Tie2. Claudin-5 at tight junctions also had a mosaic pattern, whereas VE-cadherin at adherens junctions bordered all endothelial cells. Importantly, the amounts of Tie2-pY992 and claudin-5 in aortic endothelial cells correlated with downstream polarization of VE-PTP. VE-PTP and Tie2-pY992 also had mosaic patterns in the vena cava, but claudin-5 was nearly absent and extravasated IgG was ubiquitous. Correlation of Tie2-pY992 and claudin-5 with VE-PTP polarization supports their collective interaction in the regulation of endothelial barrier function in the aorta, yet differences between the aorta and vena cava indicate additional flow-related determinants of permeability. Together, the results highlight new levels of endothelial cell functional mosaicism in the aorta and vena cava, where blood flow dynamics are well known to be heterogeneous., (© 2023. The Author(s).)

Although total arch replacement would be performed in a patient with acute type A aortic dissection and concomitant aortic aneurysm in the distal aortic arch, total arch replacement may be too invasive in elderly patients with significant morbidities. A 92-year-old female with acute type II DeBakey aortic dissection and concomitant distal aortic arch aneurysm was successfully treated with hemi-arch replacement followed by thoracic endovascular aortic repair. Hybrid two-stage repair of DeBakey type II aortic dissection complicated by distal arch aneurysm using thoracic endovascular aortic repair after hemi-arch replacement may be effective., Competing Interests: The authors declare that they have no conflicts of interest., (Published by Oxford University Press and JSCR Publishing Ltd. © The Author(s) 2023.)

Stenotrophomonas maltophilia is a Gram-negative bacterium, usually considered a nosocomial pathogen. Its role in community-acquired infections has been reported, but it is still not typically included in differential diagnoses of patients not exposed to the healthcare system. Recently, some reports suggested that liver diseases might also act as a possible risk factor for community-acquired S. maltophilia bloodstream infection. We report a case of a 77-year-old woman with a history of cirrhosis who was diagnosed with community-acquired S. maltophilia bloodstream infection. S. maltophilia not only causes hospital-acquired infections but is also emerging as a pathogen in community settings. Although community-onset infection is still rare and might have lower mortality, this antibiotic-resistant bacterial species should be considered a possible pathogen in patients with liver cirrhosis. Although trimethoprim-sulfamethoxazole is considered the first-line treatment, a study in vitro and a 4-year review of S. maltophilia susceptibility in our institution found that the bacteria were more susceptible to minocycline than to trimethoprim-sulfamethoxazole. Therefore, minocycline might become the first-line treatment in the future., Competing Interests: The authors declare that they have no competing interests., (© 2023 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd.)

The party balloon inflation maneuver increases intrathoracic pressure, decreases venous return, and after release enhances venous return to the right atrium more effectively than does the conventional Valsalva maneuver. Therefore, it shows potential for more effective detection of right-to-left shunts in patients with a patent foramen ovale. ( Level of Difficulty: Intermediate. )., Competing Interests: The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2023 The Authors.)

Vascular endothelial protein tyrosine phosphatase (VE-PTP) influences endothelial barrier function by regulating the activation of tyrosine kinase receptor Tie2. We determined whether this action is linked to the development of atherosclerosis by examining the influence of arterial shear stress on VE-PTP, Tie2 activation, plasma leakage, and atherogenesis. We found that exposure to high average shear stress led to downstream polarization and endocytosis of VE-PTP accompanied by Tie2 activation at cell junctions. In aortic regions with disturbed flow, VE-PTP was not redistributed away from Tie2. Endothelial cells exposed to high shear stress had greater Tie2 activation and less macromolecular permeability than regions with disturbed flow. Deleting endothelial VE-PTP in VE-PTP iECKO mice increased Tie2 activation and reduced plasma leakage in atheroprone regions. ApoE -/- mice bred with VE-PTP iECKO mice had less plasma leakage and fewer atheromas on a high-fat diet. Pharmacologic inhibition of VE-PTP by AKB-9785 had similar anti-atherogenic effects. Together, the findings identify VE-PTP as a novel target for suppression of atherosclerosis., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

In the initial process of coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects respiratory epithelial cells and then transfers to other organs the blood vessels. It is believed that SARS-CoV-2 can pass the vascular wall by altering the endothelial barrier using an unknown mechanism. In this study, we investigated the effect of SARS-CoV-2 on the endothelial barrier using an airway-on-a-chip that mimics respiratory organs and found that SARS-CoV-2 produced from infected epithelial cells disrupts the barrier by decreasing Claudin-5 (CLDN5), a tight junction protein, and disrupting vascular endothelial cadherin-mediated adherens junctions. Consistently, the gene and protein expression levels of CLDN5 in the lungs of a patient with COVID-19 were decreased. CLDN5 overexpression or Fluvastatin treatment rescued the SARS-CoV-2-induced respiratory endothelial barrier disruption. We concluded that the down-regulation of CLDN5 expression is a pivotal mechanism for SARS-CoV-2-induced endothelial barrier disruption in respiratory organs and that inducing CLDN5 expression is a therapeutic strategy against COVID-19.

Claudin-5 (CLDN-5) is an essential component of the tight junction seal in the blood-brain barrier. Previously, we showed that CLDN-5 modulation in vitro via an anti-CLDN-5 monoclonal antibody (mAb) may be useful for increasing the permeability of the blood-brain barrier for drug delivery to the brain. Based on these findings, here we examined the safety and efficacy of the anti-CLDN-5 mAb in a non-human primate. Cynomolgus monkeys were intravenously administered the anti-CLDN-5 mAb followed by fluorescein dye (376 Da), and the concentrations of the dye in the cerebrospinal fluid was examined. When the mAb was administered at 3.0 mg/kg, the concentration of dye in the cerebrospinal fluid was increased, and no behavioral changes or changes in plasma biomarkers for inflammation or liver or kidney injury were observed. However, a monkey that received the mAb at 6 mg/kg experienced convulsions, and subsequent histopathological examination of this animal revealed vasodilation in the liver, lung, and kidney; hemorrhage in the lung; and edema in the brain. Together, our data indicate that CLDN-5 might be a potential target for enhancing drug delivery to the brain, but also that the therapeutic window of the anti-CLDN-5 mAb may be narrow for separating efficacy and toxicity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Roundabout guidance receptor 4 (Robo4) is an endothelial-specific membrane protein that suppresses pathological angiogenesis and vascular hyperpermeability by stabilizing endothelial cells. Robo4 suppresses severe systemic inflammation induced by pathogens and endotoxins and inhibits tumor growth and metastasis, therefore serving as a potential therapeutic target. Although the regulation of Robo4 expression through transcription factors and epigenetic mechanisms has been studied, the role of histone deacetylases (HDACs) has not been explored. In the present study, we investigated the involvement of HDACs in the regulation of Robo4 expression. An HDAC inhibitor, MS-275, which inhibits HDAC1, HDAC2, and HDAC3, was found to suppress Robo4 expression in endothelial cells. Small interfering RNA (siRNA)-mediated knockdown of HDAC3, but not of HDAC1 and 2, also decreased its expression level. MS-275 downregulated the expression of the transcription factor complex GABP, in addition to suppressing Robo4 promoter activity. GABP expression was also downregulated by the siRNA against HDAC3. MS-275 decreased the transendothelial electrical resistance of a monolayer of mouse endothelial cells and increased the rate of leakage of Evans blue dye in the mouse lungs. In addition, MS-275 accelerated cell migration through the endothelial cell monolayer and augmented cell extravasation in the mouse lungs. Taken together, we demonstrated that MS-275 suppresses Robo4 expression by inhibiting HDAC3 in endothelial cells and enhances endothelial and vascular permeability. Thus, we demonstrated a novel mechanism regulating Robo4 expression and vascular permeability, which is anticipated to contribute to future therapies for infectious and inflammatory diseases.

Vascular permeability is regulated mainly by the endothelial barrier and controls vascular homeostasis, proper vessel development, and immune cell trafficking. Several molecules are involved in regulating endothelial barrier function. Roundabout 4 (Robo4) is a single-pass transmembrane protein that is specifically expressed in vascular endothelial cells. Robo4 is an important regulator of vascular leakage and angiogenesis, especially under pathological conditions. The role of Robo4 in preventing vascular leakage has been studied in various disease models, including animal models of retinopathy, tumors, diabetes, and endotoxemia. The involvement of Robo4 in vascular endothelial growth factor and inflammation-mediated signaling pathways has been well studied, and recent evidence suggests that Robo4 modulates endothelial barrier function via distinct mechanisms. In this review, we discuss the role of Robo4 in endothelial barrier function and the underlying molecular mechanisms.

Roundabout4 (Robo4) is an endothelial cell-specific protein that stabilizes the vasculature in pathological angiogenesis and inflammation. We previously determined a 3-kb Robo4 promoter and demonstrated the importance of the upstream region for nuclear factor-kappaB (NF-κB)-mediated promoter activation induced by tumor necrosis factor α (TNFα). This region contains unique genomic features, including promoter region-specific DNA hypermethylation and chromatin condensation; however, the function of the region remains poorly understood. In this study, we analyzed the DNA sequences of the region and identified a motif for polycomb repressive complex 2 (PRC2). Chromatin immunoprecipitation assay indicates the binding of the PRC2 component, SUZ12, to the motif. A mutation in the motif decreased DNA methylation in embryonic stem cells and increased Robo4 promoter activity in endothelial cells. An inhibitor for the PRC2 component, EZH2, induced the promoter activity and expression of Robo4 in endothelial cells treated with or without TNFα. Taken together, these results indicate that the PRC2 components maintain DNA hypermethylation and suppress Robo4 expression via the PRC2 binding motif in the upstream promoter.

Claudin (CLDN) proteins, a tetra-transmembrane family containing over 20 members, have been identified as key structural and functional components of intercellular seals, tight junctions (TJs). CLDNs are involved in the barrier and fence functions of TJs. Loosening the TJ barrier is one strategy for increasing drug absorption and delivery to the brain. Due to aberrant CLDN expression, the TJ fence function is frequently dysregulated in carcinogenesis. In addition, CLDN-1 is a co-receptor for the hepatitis C virus. Together these characteristics indicate CLDNs as promising targets for drug development, and CLDN binders are potential candidates for delivering drugs, treating cancer, and preventing viral infection. Before 2008, a receptor-binding fragment of Clostridium perfringens enterotoxin was the only CLDN binder available. Since then, several challenges regarding the generation of monoclonal antibodies against CLDNs have been surmounted, leading to breakthroughs in CLDN-targeted drug development. Here, we provide an overview of the recent progress in technology using created CLDN binders-anti-CLDN monoclonal antibodies., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Roundabout guidance receptor 4 (Robo4) is an endothelial cell-specific receptor that stabilizes the vasculature in pathological angiogenesis. Although Robo4 has been shown to suppress vascular hyperpermeability induced by vascular endothelial growth factor (VEGF) in angiogenesis, the role of Robo4 in inflammation is poorly understood. In this study, we investigated the role of Robo4 in vascular hyperpermeability during inflammation. Endotoxemia models using Robo4 -/- mice showed increased mortality and vascular leakage. In endothelial cells, Robo4 suppressed tumor necrosis factor α (TNFα)-induced hyperpermeability by stabilizing VE-cadherin at cell junctions, and deletion assays revealed that the C-terminus of Robo4 was involved in this suppression. Through binding and localization assays, we demonstrated that in endothelial cells, Robo4 binds to TNF receptor-associated factor 7 (TRAF7) through interaction with the C-terminus of Robo4. Gain- and loss-of-function studies of TRAF7 with or without Robo4 expression showed that TRAF7 is required for Robo4-mediated suppression of hyperpermeability. Taken together, our results demonstrate that the Robo4-TRAF7 complex is a novel negative regulator of inflammatory hyperpermeability. We propose this complex as a potential future target for protection against inflammatory diseases., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The production of antibodies against the extracellular regions (ECR) of multispanning membrane proteins is notoriously difficult because of the low productivity and immunogenicity of membrane proteins due to their complex structure and highly conserved sequences among species. Here, we introduce a new method to generate ECR-binding antibodies utilizing engineered liposomal immunogen prepared using a wheat cell-free protein synthesis system. We used claudin-5 (CLDN-5) as the target antigen, which is a notoriously difficult to produce and poorly immunogenic membrane protein with two highly conserved extracellular loops. We drastically improved the productivity of CLDN-5 in the cell-free system after suppressing and normalizing mRNA GC content. To overcome its low immunogenicity, two engineered antigens were designed and synthesized as proteoliposomes: a human/mouse chimeric CLDN-5, and a CLDN-5-based artificial membrane protein consisting of symmetrically arranged ECRs. Intraperitoneal immunization of both engineered CLDN-5 ECR antigens induced ECR-binding antibodies in mice with a high success rate. We isolated five monoclonal antibodies that specifically recognized CLDN-5 ECR. Antibody clone 2B12 showed high affinity (<10 nM) and inhibited CLDN-5-containing tight junctions. These results demonstrate the effectiveness of the methods for monoclonal antibody development targeting difficult-to-produce membrane proteins such as CLDNs.

Although transcription factors regulating endothelial cell (EC)-specific gene expression have been identified, it is not known how those factors induce EC-specificity. We previously reported that DNA hypomethylation of the proximal promoter elicits EC-specific expression of Roundabout4 (Robo4). However, the mechanisms establishing EC-specific hypomethylation of the Robo4 promoter remain unknown. In this study, we demonstrated that the hypermethylated Robo4 proximal promoter is demethylated as human iPS cells differentiate into endothelial cells. Reporter assays demonstrated that ETV2, an ETS family transcription factor, bound to ETS motifs in the proximal promoter and activated Robo4 expression. Immunoprecipitation demonstrated direct interaction between ETV2 and methylcytosine-converting enzymes TET1 and TET2. Adenoviral expression of ETV2-TET1/TET2 complexes demethylated the Robo4 promoter and induced Robo4 expression in non-ECs. In summary, we propose a novel regulatory model of EC-specific gene expression via promoter demethylation induced by ETV2-TET1/TET2 complexes during endothelial differentiation.

Roundabout4 (Robo4) is an endothelial cell-specific receptor that stabilizes vasculature in pathological angiogenesis. Previous studies have shown that Robo4 is a potential therapeutic target for inflammatory diseases, but its precise roles in inflammation remain unclear. To investigate physiological Robo4 functions in inflammation, we performed a loss-of-function study in vitro and in vivo using lipopolysaccharide (LPS)-induced endotoxemia models. Subcutaneous injection of LPS into Robo4-knockout mice reduced circulating IL-6 levels. siRNA-mediated Robo4 knockdown suppressed IL-6 production induced by LPS, IL-1β, and TNFα, in human umbilical vein endothelial cells (HUVECs). Coculture experiments with HUVECs and a monocytic cell line, U937 cells, demonstrated that Robo4 knockdown suppresses IL-6 production by both endothelial cells and U937 cells. Further coculture experiments demonstrated that Robo4 knockdown inhibited a novel IL-6 amplification mechanism mediated by crosstalk between endothelial cells and U937 cells via direct interactions and two mediators, GM-CSF and IL-1β. Taken together, we demonstrated novel Robo4 functions in inflammation, i.e., it promotes IL-6 production by endothelial cells and immune cells via crosstalk., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A current bottleneck in the development of central nervous system (CNS) drugs is the lack of drug delivery systems targeting the CNS. The intercellular space between endothelial cells of the blood-brain barrier (BBB) is sealed by complex protein-based structures called tight junctions (TJs). Claudin-5 (CLDN-5), a tetra-transmembrane protein is a key component of the TJ seal that prevents the paracellular diffusion of drugs into the CNS. In the present study, to investigate whether CLDN-5 binders can be used for delivery of drugs to the CNS, we generated monoclonal antibodies (mAbs) specific to the extracellular domains of CLDN-5. In an in vitro model of the BBB, the anti-CLDN-5 mAbs attenuated trans-epithelial/endothelial electrical resistance and enhanced solute permeation. These anti-CLDN-5 mAbs are potential leads for the development of novel drug delivery systems targeting the CNS., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Objectives: The management of dysphagia requires a multidisciplinary approach, especially in large-scale hospitals. We introduce a novel protocol using a Wi-Fi-based flexible endoscopic evaluation of swallowing (FEES) system and aim to verify its effectiveness in evaluation and rehabilitation of inpatients with dysphagia., Method: We conducted novel Wi-Fi-based FEES at the bedside using 3 iPads as monitors and recorders. Functional outcomes of swallowing in 2 different hospitals for acute care with conventional wired or wireless FEES were compared retrospectively., Results: Using the wireless system, we could visit more patients in a short period of time. Furthermore, a large multidisciplinary team was able to be present at the bedside, which made it easy to hold discussions and rapidly devise appropriate rehabilitation strategies. Aspiration pneumonia recurred in a few cases following our intervention with wireless FEES. Functional oral intake score was significantly increased following the intervention. Moreover, the number of deaths during hospitalization using wireless FEES evaluation was lower than those observed using the conventional system., Conclusion: Wi-Fi-based wireless FEES system, the first of its kind, allowed our multidisciplinary team to easily and effectively assess inpatients with dysphagia by facilitating simple examinations and intensive transprofessional discussions for patient rehabilitation., (© The Author(s) 2016.)

Roundabout4 (Robo4) is an endothelial cell-specific receptor that regulates vascular stability. Recently, Robo4 has been shown to regulate vascular permeability in inflammation. However, the mechanisms regulating the Robo4 gene in the context of inflammation are poorly understood. In this study, we found that intravenous injection of tumor necrosis factor (TNF) α increased Robo4 expression in mouse organs. In vitro analyses showed that TNFα increased Robo4 expression in human primary endothelial cells, but not in cells pretreated with a nuclear factor (NF)-κB inhibitor. Reporter assays using wild-type and mutant Robo4 promoters indicated that TNFα activated the Robo4 promoter and that both the -2753 and -2220 NF-κB motifs were essential for this activation. Electrophoretic mobility shift assays demonstrated that the NF-κB p65-p50 heterodimer bound to these motifs. These findings were further supported by chromatin immunoprecipitation assays in endothelial cells. Taken together, these results indicated that TNFα induced Robo4 expression by facilitating NF-κB p65-p50 heterodimer binding to the -2753 and -2220 motifs in the Robo4 promoter in endothelial cells in the context of inflammation.

[Purpose] An earlier study divided reaching activity into characteristic phases based on hand velocity profiles. By synchronizing muscle activities and the acceleration profile, a phasing approach for reaching movement, based on hand acceleration profiles, was attempted in order to elucidate the roles of individual muscle activities in the different phases of the acceleration profile in reaching movements. [Subjects and Methods] Ten healthy volunteer subjects participated in this study. The aim was to electromyographically evaluate muscles around the shoulder, the upper trapezius, the anterior deltoid, the biceps brachii, and the triceps brachii, most of which have been used to evaluate arm motion, as well as the acceleration of the upper limb during simple reaching movement in the reach-to-grasp task. [Results] Analysis showed the kinematic trajectories of the acceleration during a simple biphasic profile of the reaching movement could be divided into four phases: increasing acceleration (IA), decreasing acceleration (DA), increasing deceleration (ID), and decreasing deceleration (DD). Muscles around the shoulder showed different activity patterns, which were closely associated with these acceleration phases. [Conclusion] These results suggest the important role of the four phases, derived from the acceleration trajectory, in the elucidation of the muscular mechanisms which regulate and coordinate the muscles around the shoulder in reaching movements.

Background: Most people with rheumatoid arthritis (RA) are physically inactive. An accelerometer worn on the waist has been used to evaluate physical activity in people with chronic conditions. It is useful for evaluating moderate to vigorous activity, although it tends to underestimate light or mild activities such as housework or family duties. An accelerometer worn on the wrist (i.e., actigraph) has recently been used to capture daily physical activity in inactive individuals. The purposes of this study were to investigate physical activity measured by an actigraph in patients with RA and in healthy individuals and to investigate the association between actigraphic data and self-reported physical function., Methods: The subjects were 20 RA patients and 20 healthy individuals. All participants wore an actigraph on their wrist for 6-7 consecutive days. They also completed the Health Assessment Questionnaire disability index (HAQ-DI) and the Medical Outcomes Study (MOS) 36-item short form health survey (SF-36). We extracted three parameters from the actigraphic data: mean activity count (MAC), peak activity count (PAC), and low activity ratio (LAR). These three parameters were compared between the RA patients and healthy individuals and with the self-reported questionnaires., Results: The MAC was significantly lower and the LAR was significantly higher in RA patients than in healthy individuals. The PAC was not different between the two groups. The LAR was negatively correlated with the MAC for the RA patients and for the healthy individuals. The decrease ratio of the LAR with the increase of the MAC for the RA patients was twice that of the healthy participants. In the RA patients, the LAR was significantly and moderately correlated with the HAQ-DI score and two dimensions of the SF-36 (i.e., "physical functioning" and "bodily pain")., Conclusion: Investigation of the proportion of low activity count using an actigraph may be useful to identify characteristics of the physical function in RA patients.

Background: While trunk movement accompanies arm elevation, trunk muscle activities during arm elevation at different speeds are unclear. The purpose of this study was to examine the trunk muscle activities at various speeds of arm elevation and then to evaluate their roles in trunk kinematics., Methods: Twenty-two healthy subjects participated. The participants performed right shoulder flexion at three different speeds. Surface electromyography was used to measure the activities of bilateral external oblique muscles (EO), internal oblique muscles (IO), rectus abdominis muscles (RA) and lumber erector spinae muscles (ES). A three-dimensional motion analyzer was used to measure arm and trunk movements., Results: In natural and slow movements, the muscle activities of left ES, right EO and left IO were significantly augmented compared with those of the contralateral muscles, in the relatively late phase. In fast movement, the muscle activities of both ES were significantly augmented during the early phase compared with later in the action. The muscle activities of the left ES and the right EO were significantly augmented compared with those of the contralateral muscles. There was a consistent pattern of trunk extension, lateral flexion and rotation during arm elevation, irrespective of the speed., Conclusions: Bilateral ES activity may be required for back-extension torques, especially for the early phase of rapid elevating motion. The anterior muscles' activity may contribute to the production of anterior force against the backward movement of the center of mass of the upper limb in the late phase. Trunk rotation, controlled by the trunk muscles in harmony, may assist the scapular movement to align the scapular plane in the arm elevating plane.

A prospective radiographic study.The purpose of this study was to analyze whether a novel skull clamp positioning system and technique is useful for obtaining good, quantitative cervical sagittal alignment during posterior cervical surgery.Different surgical procedures depend on cervical spine positioning. However, maneuver of the device and cervical position depends on the skill of the operator.This study included 21 male and 10 female patients with cervical spondylotic myelopathy and ossification of the posterior longitudinal ligament of the cervical spine, undergoing posterior cervical surgery using the novel skull clamp positioning system. The average patient age was 68.6 years (range: 56-87 years). The novel system has a scale to adjust the neck position and to enable intended cervical sagittal alignment. First, the patient was placed on the operating table in the prone position with preplanned head-neck sagittal alignment (neutral position in general). The head was rotated sagittally, and the head was positioned in the military tuck position with the novel device that was used to widen the interlaminar space. After completing the decompression procedure, the head was rotated again back to the initial preplanned position. During this position change, the scale equipped with the device was useful in determining accurate positions. The C0-C1, C0-C2, C1-C2, C2-C7, and C0-C7 angles were measured on lateral radiographs taken pre-, intra-, and postoperatively.This novel system allowed us to obtain adequate, quantitative cervical sagittal alignment during posterior cervical surgery. There were no clinically significant differences observed between the pre- and postoperative angles for C1-C2 and C2-C7.Sagittal neck position was quantitatively changed during posterior cervical surgery using a novel skull clamp positioning system, enabling adequate final cervical sagittal alignment identical to the preplanned neck position.

[Purpose] The purpose of this study was to clarify the relationship between acceleration and joint movement by synchronizing accelerometers and a three-dimensional motion analysis system, and to show the utility of an accelerometer as a postural control assessment tool. [Subjects and Methods] Head, lumbar, shank accelerations and various joint angles during single-leg standing were measured of 20 healthy males. Root mean squares of acceleration and joint angle were calculated. Fast Fourier transform analysis was performed for head, lumbar, and shank accelerations, and the median frequencies were calculated. Then, principal component analysis was performed for the median frequency of each acceleration. Stepwise multiple regression analysis was also used to examine the relationship between joint angle and acceleration. [Results] The score of the first principal component was highest for shank acceleration, while that of the second principal component was highest for lumbar and head accelerations. In multiple regression analysis, hip flexion/extension and adduction/abduction were identified as variables associated with head acceleration. [Conclusion] We were able to confirm the aggregation of acceleration into two components, which we interpreted as postural control strategies using primarily the ankle and hip joints. Furthermore, though multiple regression analysis, we were able to clarify the joint movement indicated by acceleration of each segment.